The regulation of cardiac and skeletal muscle contraction by Ca++ involves a series of complex protein-protein interactions between the three subunits (TnT, the tropomyosin (Tm) binding subunit, TnI, the inhibitory subunit, and TnC, the Ca++ binding subunit) of troponin (Tn), the two subunits of tropamyosin and actin. Quite a bit is known about the subunit interactions between the isolated Tn component pairs, and this information has been extrapolated back to the intact complex, although there is no easy way to test the validity of these extrapolations. One way we propose to study these interactions and their role in muscle regulation is to use monoclonal antibodies as specific probes of these protein-protein interactions. The basic strategy in this project will be to initially prepare monoclonal antibodies against the three individual Tn subunits (and selected fragments of the subunits) and then characterize them with regards to their specificity, stoichiometry, and affinity. We will also determine as nearly as possible the location of the various antigenic determinants. Since the amino acid sequence of all of the Tn subunits is known and a variety of proteolytic fragments are available from them, it should be possible to narrow down the regions of the proteins which make up the antigenic determinant using a variety of techniques. We will also determine whether or not these different antibodies can bind to these antigenic sites on the individual proteins in the various reconstituted systems (e.g. TnC-TnI, TnI-TnT, TnT-TnC, Tn, Tn.Tm, Tn.Tm.Actin, etc.) in different conformational states (e.g., Ca++, extent of filament overlap, rigor, etc.) We will also test to see if any of these antibodies affect the function of Tn by testing them against myofibrils (ATPase assays) and skinned fibers (tension development). In this way it should be possible to map regions of the various Tn subunits that are functionally important in addition to those regions which are/are not available for antibody binding in the different protein complexes and/or conformational states. Other groups (1, 2) have attempted to ask similar questions about Tn subunit interactions through the use of either protein fragments or chemical reactivity studies, methods which probably substantially alter the native structure of the proteins and, in addition, only give information about the interactions and not about function. In contrast, the approach designed in this application should yield information about Tn subunit interactions in the native state as well as their function.